Complexes between synthetic polymers and biological macromolecules can provide important commercial therapeutics as well as valuable building blocks of structured materials and sensors. For example, conjugation of therapeutic proteins with polymers, such as with polyethylene glycol, has been shown to prolong the serum half-life and reduce immunogenicity of the proteins. Conjugates of biomacromolecules covalently linked to synthetic polymers at the ends of the synthetic polymers are examples of such complexes. In this instance, controlling the site of covalent conjugation on the biomacromolecule, the number of polymer chains conjugated, and the length, dispersity, and architecture of the synthetic polymer chains are particularly critical to resultant properties.
The currently used method to prepare covalent biomacromolecule-polymer complexes involves first preparing a polymer chain modified with a reactive end group and subsequently conjugating that preformed polymer to the biomacromolecule which contains either a natural or a non-natural amino acid. Kiick, K. L. et al. (“Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase Activity of a Bacterial Expression Host”, Angew. Chem., Int. Ed., 39 (2000) p2148-2152; “Identification of an Expanded Set of Translationally Active Methionine Analogues in Escherichia Coli”, FEBS Lett., 502 (2001) p25-30) discusses the incorporation of non-natural amino acids into proteins.
U.S. Pat. No. 5,998,588 to Hoffman et al. is one example of several patents issued to Hoffman covering various procedures for the conjugation of preformed polymer chains to numerous biomolecules including proteins.
Kochendoerfer, G. G. et al. (“Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis Protein”, Science, 299 (2003) p884-887) gives an example of the formation of a polymer modified protein by chemical synthesis, an amino-oxy group on the polymer being linked at ketone bearing Lys (N-levulinyl) residues on the peptide.
Wang Y. et al. (“Structural and Biological Characterization of Pegylated Recombinant Interferon Alpha-2b and its Therapeutic Implications”, Adv. Drug Delivery Rev., 54 (2002) p547-570) discusses the therapeutic use of small proteins (type 1 interferon alpha) as anti-infectives and anti-tumor agents. However, the utility of such therapy is limited by the half-life of interferon and its rapid clearance from the body. The efficacy of interferon can be improved (i.e., converted to a long acting agent) by reacting the protein with mono-methoxy polyethylene glycol to form pegylated interferon (PEG Intron®), a covalent conjugate of IFN-α2b linked to a 12,000 Da PEG molecule. Pegylation occurs at any or all of numerous nucleophilic sites in the protein (the s-amino groups of the 10 lysines, the α-amino group at the N-terminal cysteine, the imidazolyl nitrogens of the three histidines and the hydroxyl groups at the 14 serine, 10 threonines, and 5 tyrosines). Because of the numerous potential reaction sites, a heterogeneous mixture of various different modified proteins is produced.
Kinstler, O. (“Mono-N-terminal Poly(ethylene glycol)—Protein Conjugates”, Adv. Drug Delivery Rev., 54, (2002) p477-485) also reports on the formation of PEG-protein conjugates. They maximize the selectivity of the PEG aldehyde conjugation to the N-terminus of an unprotected polypeptide chain by taking advantage of the differences between pKa values of the α-amino group of the N-terminal amino acid residue and the ε amino group of the Lys residues in the peptide backbone.
Another approach is to target cysteine thiols using a polymer (PEG) activated with maleimides, vinyl sulfones, pyridyl disulfides, or other compounds reactive to thiols, thus taking advantage of the scarcity of free cysteines in proteins. Chapman, A. P., et al. (“Therapeutic Antibody Fragments with Prolonged In Vivo Half-Lives”, Nat. Biotechnol, 17 (1999) p780-783) (referenced in Kinstler et al.).
The state of the art regarding polymeric drugs, polymer-drug conjugates, polymer-protein conjugates, polymeric micelles with covalently bound drugs and multi-component complexes is reviewed by Duncan, R. (“The Dawning Era of Polymer Therapeutics”, Nat. Rev. Drug Discovery, 2, (May 2003) p347-360). Described are conjugates formed by reacting the polymer with the biomolecule. The polymeric materials identified include PEG, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, poly(vinylpyrrolidone) (PVP), poly(ethyleneimine) (PEI), polyamidoamines (DIVEMA), natural polymers including dextran, hyaluronic acid, chitosans and synthetic polyamino acids such as poly(L-lysine), poly(glutamic acid), poly(malic acid) and poly(aspartamides).
Hannink, J. M. (“Protein-Polymer Hybrid Amphiphiles”, Anew. Chem., Int. Ed., 40, (2001) p4732-4734) discloses the irreversible association of two molecules of monobiotinylated polymers with streptavidin to form an amphiphilic protein-polymer hybrid.
Unfortunately, synthesis of the polymers with reactive end groups and separation of the excess (unreacted) polymer chains from the conjugate formed between the polymer and biomolecule is difficult and time consuming. In addition, many of these methods are not quantitative or specific and do not allow for control over the placement and number of polymer chains. To make these conjugates available, a simple and effective preparation of biomacromolecule-polymer complexes is needed.